Morishita R, Gibbons G H, Kaneda Y, Ogihara T, Dzau V J
Falk Cardiovascular Research Center, Stanford University School of Medicine, CA 94305-5246.
Gene. 1994 Nov 4;149(1):13-9. doi: 10.1016/0378-1119(94)90406-5.
Using a highly efficient viral HVJ (hemagglutinating virus of Japan) liposome-mediated transfer method, we examined the cellular fate of antisense oligodeoxyribonucleotides (oligos) in the vessel wall in vivo. Direct transfer of unmodified FITC (fluorescein isothiocyanate)-labeled oligos into injured rat carotid arteries showed, localized in the medial layer, fluorescence that disappeared within 1 day. In contrast, transfection of unmodified FITC-oligos by the HVJ-liposome method showed, concentrated in the medial layer, high levels of fluorescence that were sustained for at least 1 week. Moreover, we demonstrated nuclear localization and accumulation of fluorescence in the vessel wall using this method. To examine the therapeutic utility of this method, we transferred antisense phosphorothioate oligos against cyclin B1- and CDC2 kinase-encoding genes into balloon-injured rat carotid artery as a potential therapy for experimental restenosis. Two weeks after transfection, antisense oligo treatment directed against either CDC2 kinase or cyclin B1 resulted in a partial, but significant, inhibition in neointima formation. In contrast, transfection of either sense or scrambled control oligos had no effect. Interestingly, co-transfection of antisense oligos against CDC2 kinase and cyclin B resulted in further inhibition of neointima formation, as compared to blockade of either gene target alone. These results demonstrate that: (i) the HVJ-liposome method enhances the half life and nuclear localization of antisense oligos in the vessel wall in vivo; and (ii) HVJ-mediated administration of antisense CDC2 kinase and cyclin B1 oligos produces a sustained inhibition of neointima formation after balloon angioplasty.
我们使用高效的病毒HVJ（日本血凝病毒）脂质体介导的转移方法，在体内研究了血管壁中反义寡脱氧核糖核苷酸（oligos）的细胞命运。将未修饰的异硫氰酸荧光素（FITC）标记的oligos直接转移到损伤的大鼠颈动脉中，结果显示荧光位于中层，1天内消失。相比之下，通过HVJ-脂质体方法转染未修饰的FITC-oligos，结果显示荧光高度集中在中层，且持续至少1周。此外，我们使用该方法证明了血管壁中荧光的核定位和积累。为了研究该方法的治疗效用，我们将针对细胞周期蛋白B1和CDC2激酶编码基因的反义硫代磷酸酯oligos转移到球囊损伤的大鼠颈动脉中，作为实验性再狭窄的潜在治疗方法。转染后两周，针对CDC2激酶或细胞周期蛋白B1的反义oligo治疗导致内膜增生受到部分但显著的抑制。相比之下，转染正义或随机对照oligos则没有效果。有趣的是，与单独阻断任何一个基因靶点相比，同时转染针对CDC2激酶和细胞周期蛋白B的反义oligos导致内膜增生受到进一步抑制。这些结果表明：（i）HVJ-脂质体方法可提高反义oligos在体内血管壁中的半衰期和核定位；（ii）HVJ介导的反义CDC2激酶和细胞周期蛋白B1 oligos给药可在球囊血管成形术后持续抑制内膜增生。
